U.S. patent number 5,426,030 [Application Number 07/572,875] was granted by the patent office on 1995-06-20 for apparatus for determination of hdl cholesterol.
This patent grant is currently assigned to Boehringer Mannheim GmbH. Invention is credited to Klaus Buecker, Ulfert Deneke, Uwe Goebbert, Gerhard Hiller, Hartmut Merdes, Walter Rittersdorf.
United States Patent |
5,426,030 |
Rittersdorf , et
al. |
June 20, 1995 |
Apparatus for determination of HDL cholesterol
Abstract
The invention relates to an apparatus and a method useful in
separating non-high density lipoproteins, referred to as
"non-HDLs", from biological fluids containing them. A porous
carrier is provided which contains a non-HDL precipitating agent.
One need only contact the sample of interest to the carrier for one
minute or less, after which the precipitated non-HDLs are no longer
present in the sample being tested. The applications of the method
include the ability to determine high density lipoproteins in the
sample without interference from non-HDLs.
Inventors: |
Rittersdorf; Walter (Mannheim,
DE), Deneke; Ulfert (Rimbach-Zotzenbach,
DE), Hiller; Gerhard (Mannheim, DE),
Merdes; Hartmut (Heidelberg, DE), Buecker; Klaus
(Viernheim, DE), Goebbert; Uwe (Mannheim,
DE) |
Assignee: |
Boehringer Mannheim GmbH
(Mannheim, DE)
|
Family
ID: |
6388410 |
Appl.
No.: |
07/572,875 |
Filed: |
August 24, 1990 |
Foreign Application Priority Data
|
|
|
|
|
Sep 1, 1989 [DE] |
|
|
39 29 032.8 |
|
Current U.S.
Class: |
435/11; 422/401;
436/170; 436/71 |
Current CPC
Class: |
C12Q
1/60 (20130101); G01N 33/526 (20130101); G01N
33/92 (20130101); Y10T 436/255 (20150115); Y10T
436/25125 (20150115); Y10T 436/104165 (20150115) |
Current International
Class: |
C12Q
1/60 (20060101); G01N 33/52 (20060101); G01N
33/92 (20060101); C12Q 001/60 (); G01N 021/00 ();
G01N 033/92 () |
Field of
Search: |
;435/11,7.8,7.1
;436/170,71 ;422/56,57 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Weber, Walter J. Physicochemical Processed Wiley-Interscience N.Y.
1972 p. 131. .
Derwent Abstract 89-061491. .
Derwent Abstract 90-295783. .
Bachorik et al., Meth. Enzymol 129: 78-100 (2986)..
|
Primary Examiner: Beisner; William H.
Assistant Examiner: Gitomer; Ralph
Attorney, Agent or Firm: Felfe & Lynch
Claims
We claim:
1. Apparatus for determining high density lipoprotein (HDL)
cholesterol in a lipoprotein containing body fluid sample,
comprising:
(i) a sample application layer,
(ii) a porous carrier layer containing a precipitating agent which
precipitates non high density lipoproteins (non HDLs) but not HDL,
wherein said porous carrier layer is in fluid contact with said
sample application layer,
(iii) a fluid transport means, a first portion of which is in fluid
contact with said porous carrier layer, a second portion of which
extends away from said porous carrier layer,
(iv) a support means attached to said fluid transport means,
and
(v) a reagent layer which contains a cholesterol determining
reagent, wherein said reagent layer is attached to said support
means by a connecting means which permits contact of said reagent
layer to said second portion of said fluid transport means upon
application of pressure to said reagent layer.
2. The apparatus of claim 1, wherein said porous carrier layer
comprises a fiber fleece having a weight per unit area of from 10
to 100 g/m.sup.2 and a water absorbing capacity of 25 to 200
g/m.sup.2 at a thickness of from 0.030 to 0.150 mm.
3. The apparatus of claim 2, wherein said fleece comprises fibers
having a diameter of from 3 to 100 um.
4. The apparatus of claim 2, wherein the fibers of said fiber
fleece comprise synthetic resin fibers, natural fibers or glass
fibers.
5. The apparatus of claim 1, wherein said precipitating agent
comprises a combination of (i) a polyanion selected from the group
consisting of phosphotungstic acid, heparin and dextran sulphate,
and (ii) a bivalent cation selected from the group consisting of
Mg.sup.2+, Mn.sup.2+, and Ca.sup.2+.
6. The apparatus of claim 5, wherein said polyanion is dextran
sulphate having a molecular weight of about 50,000 g/mol, and said
bivalent cation is Mg.sup.2+, in the form of magnesium acetate.
7. The apparatus of claim 1, wherein said porous carrier layer
comprises a hydrophilic membrane having a thickness of from 20-250
um and a pore size of from 0.2 to 20 um.
8. Apparatus for determining high density lipoprotein (HDL)
cholesterol in a lipoprotein containing body fluid sample,
comprising: (i) a sample application layer,
(ii) a first porous carrier layer containing a precipitating agent
which precipitates non high density lipoproteins (non HDLs) but not
HDL, wherein said porous carrier layer is in fluid contact with
said sample application layer,
(iii) a second porous carrier layer comprising a fiber mesh, the
fibers of which have a weight per unit area of from 20 to 50
g/cm.sup.2, and water absorbing capacity of 250-500 g/m.sup.2 at a
thickness of from 0.19 to 0.23 mm,
(iv) a fluid transport means, a first portion of which is in fluid
contact with said porous carrier layer, a second portion of which
extends away from said porous carrier layer,
(v) a support means attached to said fluid transport means, and
(vi) a reagent layer which contains a cholesterol determining
reagent, wherein said reagent layer is attached to said support
means by a connecting means which permit contact of said reagent
layer to said second portion of said fluid transport means upon
application of pressure to said reagent layer.
Description
The invention concerns a method for the quantitative determination
of HDL (High Density Lipoprotein) in biological fluids and an agent
suitable therefor.
Total cholesterol in blood, plasma or serum is one of the best
known parameters for assessing the extent of risk of a coronary
heart disease.
However, the concentration of total cholesterol is only of limited
value for the assessment of individual risk. The measurement of the
cholesterol in the lipoproteins of low density (Low Density
Lipoproteins=LDL) on the one hand and in the lipoproteins of high
density (High Density Lipoproteins=HDL) on the other hand is more
meaningful. Epidemiological and clinical studies have shown that
there is a positive correlation between LDL cholesterol and
coronary heart disease and a negative correlation between HDL
cholesterol and coronary heart disease.
As a close approximation the determination of the HDL as well as
the total cholesterol is sufficient for an assessment of risk. This
course is preferably followed at present in diagnostic
practice.
The other lipoprotein classes (LDL, VLDL, chylomicrons) which are
present have to be separated in order to determine HDL cholesterol
separately. The potential methods of separation are based on
differences in the flotation densities (sequential flotation or
equilibrium sedimentation, both in the ultracentrifuge), on
different surface charges (electrophoreses on paper or agarose as
carrier) or on differences in the apolipoproteins (immunochemical
methods using specific antibodies). All these methods of separation
are expensive, time-consuming and not established in routine
laboratories. Precipitation reactions (in Monographs on
Atherosclerosis, Vol. 11 (1982), Clackson, T. B., Kritchevsky, D.,
Pollak, O. J. eds; Lipoprotein Precipitation, Burstein, M.,
Legmann, P. and in Meth. in Enzymology, Vol. 129 (1986)) whose
specificity depends on the particle dimension and the surface
charge are cheap, relatively easy to handle and therefore
widespread. Polymeric substances serve as precipitation reagents,
and these are usually polyanionic. Polymers which are uncharged are
also suitable. The polyanions usually need bivalent cations in
order to develop their precipitating effect, while the uncharged
polymers do not require them.
The experimental procedures and the concentrations which are used
for the combined precipitation agents are designed to
quantitatively precipitate all lipoproteins except HDL, to separate
these precipitates from the liquid fraction of the sample in a
suitable manner and subsequently to quantify the HDL in the liquid
fraction of the sample by means of a cholesterol assay. For this,
depending on how the test is carried out, a defined volume of
precipitating agent (in suitable concentrations) is mixed
intensively with a defined volume of the sample to be determined.
It is the state of the art to allow a reaction time of at least 10
minutes for the precipitation and only after this time interval has
elapsed to separate sedimented non-HDL lipoprotein precipitates and
HDL remaining in the liquid fraction by centrifugation. The
centrifugation step also needs some time.
This method is much too time-consuming for a routine test. In
addition, centrifugation steps with subsequent separation of the
supernatant need complicated additional equipment and a transfer
step. The required pipetting procedure in which a defined amount of
supernatant is taken is, in addition, a source of error which can
lead to less precise measurements.
It is therefore the object of the invention to avoid the
disadvantages of the state of the art and to provide a method for
the separation of non-HDL lipoproteins from biological fluids,
which can be carried out more rapidly and without complicated
additional equipment and which allows a more rapid and a simpler
HDL cholesterol determination.
The object is achieved by a method for the separation of non-HDL
lipoproteins from biological fluids, in which the biological fluid
containing non-HDL lipoproteins is applied onto a carrier through
which liquids can flow and which contains a precipitating agent for
non-HDL lipoproteins. The invention also provides an agent for the
separation of non-HDL lipoproteins and a rapid diagnostic agent
which contains this agent.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 represents a cross-section showing the layers of the test
strip.
FIG. 2 represents a side view of the test strip.
It has been found that the precipitation of the non-HDL
lipoproteins proceeds particularly rapidly and specifically with
conventional, well-known precipitating agents when these
precipitating agents are applied in a finely dispersed form onto a
carrier through which liquids can flow and the sample has to flow
through this carrier. In general, the precipitation takes less than
1 minute. Carriers through which liquids can flow are: papers,
fabrics made of synthetic fibres such as polyester or polyamide or
others, fabrics made of natural fibres such as cotton, silk or
others or mixtures of these materials. In this connection, the
structure of the fabrics can be monofilament or multifilament,
multifilament forms being preferred.
The fibres which compose the carrier through which liquids can flow
preferably have a fibre diameter from 3 to 100 .mu.m, preferably 5
to 50 .mu.m. The carrier has, in particular, a weight per unit area
of 10 to 100 g/m.sup.2, preferably 10 to 50 g/m.sup.2 at a
thickness of 0.030 to 0.150 mm, and the ability to absorb water is
25 to 100 g/m.sup.2, preferably 40 to 70 g/m.sup.2 at the
thicknesses mentioned above.
Other suitable carriers are arrangements of glass fibres and
mixtures of glass fibres with the fibres mentioned above through
which liquids can flow--preferably in the form of fleeces--and
membranes in different forms. The membranes should have hydrophilic
properties, a thickness between 20-250 .mu.m, preferably 70-150
.mu.m and pore sizes between 0.2-20 .mu.m, preferably 5-15
.mu.m.
The transport of liquid in these carriers is based on capillary
forces.
The precipitating agents are preferably applied to the carrier by
impregnating the carrier with a solution, emulsion or suspension of
the precipitating agent and subsequent drying. In this process
other useful additives such as pH buffer substances or detergents
etc. can also be applied.
In this construction all chemical compounds are suitable as
precipitating agents which can also be used in a "wet chemical"
process for the precipitation of lipoproteins as long as they
dissolve quickly enough in the sample solution. Certain polyanions
in combination with bivalent cations are particularly well known.
Examples of these include combinations of phosphotungstic acid and
magnesium chloride, of heparin and manganese (II) chloride or of
dextran sulphate and magnesium chloride. It should be noted that,
in principle, each polyanion can be combined with each of the three
cations (Mg.sup.2+, or Mn.sup.2+, or Ca.sup.2+) which results,
however, in slight differences in their capacity to precipitate
lipoproteins (Burstein, 1986). The concentration of the chosen
cation can be adapted accordingly in order to specifically
precipitate the non-HDL lipoproteins. The molecular size of the
polyanion used also influences the capacity to precipitate
lipoproteins and should be taken into account when choosing the
concentrations. The use of dextran sulphate with a molecular weight
of 500000 and 50000, for example, is known. Both are also suitable
for an application on carriers through which liquids can flow.
Dextran sulphate with a molecular weight of 50000 combined with
Mg.sup.2+ is, however, preferred in which case Mg.sup.2+ is
preferably used in the form of magnesium acetate. In principle,
magnesium sulphate, magnesium chloride and magnesium aspartate can
also be used.
The concentration of the precipitating agent can be matched exactly
to the volume of the sample to be examined.
The non-HDL lipoproteins are precipitated by bringing a sample into
contact with the carrier through which liquids can flow and which
contains the precipitating agent. The precipitation reaction is
started thereby. The time interval during which the sample is in
contact with the precipitating agent on the precipitating agent
carrier can be adjusted in a simple manner by varying the suction
pressure by means of the interstitial volumes or the hydrophilicity
of the carrier. The achieved flow rates of the sample through the
carrier are very important for the test performance. The
precipitate formation is completed after less than one minute, at
best even after about 10 second and the liquid can be removed from
the carrier material. This can be effected continously or
discontinously, for example by decanting, using gravitational force
or by aspirating. The fluid is preferably drawn into a further
carrier by capillary forces in which the precipitated non-HDL
lipoproteins are separated. This process requires that the carriers
are in contact with one another. The separation of the precipitates
by other methods such as centrifugation is less preferable even
though this is also possible without losing the advantage of the
fast precipitation.
It was found that lipoprotein precipitates which are formed by the
action of a combination of polyanions and the bivalent cations
mentioned above on biological samples can be retained in a mesh of
fibres.
The arrangement of the fibres in the mesh is preferably disordered.
Glass fibres, cellulose fibres, polyamide fibres and polyester
fibres or mixtures thereof are preferably used as the fibres. Glass
fibres and mixtures with the fibres mentioned above are
particularly preferred. The fibres which make up the mesh
preferably have a diameter of 0.2 to 10.0 .mu.m, preferably a
diameter of 0.5 to 5.0 .mu.m. The mesh has a weight per unit area
of 20 to 50 g/m.sup.2, preferably 23 to 30 g/m.sup.2 and a capacity
to absorb water of 250 to 500 g/m.sup.2, preferably of 320 to 420
g/m.sup.2 thickness of 0.19 to 0.23 mm.
Systems containing glass fibres, their possible spatial arrangement
and the dimension of the fibres are described in detail in DE 30 29
579. Furthermore, the mesh of fibres can be hardened by the
addition of suitable binding agents, either of an inorganic nature
(e.g. water glass) or organic nature (e.g. polymers such as
polyvinyl acetate, polyacrylic acid ester or similar polymers).
These additives inter alia agglutinate the fibres at those
positions where they are in contact with one another and in this
way they improve the mechanical stability of the fibre mesh.
The liquid fraction of the applied sample spreads unhindered
through the whole mesh and it can also leave it when the geometric
arrangement is approximately arranged or configured or by
overcoming the internal capillary forces of the membrane. In this
way the separation of precipitated non-HDL lipoproteins and
non-precipitated HDL can be achieved with simple devices.
Biological fluids from which the non-HDL lipoproteins can be
separated according to the present invention include whole blood,
serum and plasma.
The method can be particularly advantageously used in methods for
the quantitative determination of HDL cholesterol on test strips.
For this, the fluid from which the non-HDL lipoproteins have been
separated according to the method described above, is brought into
contact with reagents which are necessary and/or useful for
carrying out the test reaction, for example a test reaction for
cholesterol. Such reagents are known to the expert, for example
from EP-B-0016387, and can be adapted to the expected cholesterol
concentrations (0-100 mg/dl). They can for example, also be present
in the form of a film or a coating on a porous carrier. Such
embodiments of reagent forms are known to the man skilled in the
art.
An embodiment of a method for the determination of HDL cholesterol
which contains the preferred embodiment for separating the non-HDL
lipoproteins is shown in FIG. 1. A rapid diagnostic agent (1)
contains several layers of a carrier in a casing (2).
A fibre mesh (5) with a separating capacity for lipoprotein
precipitates which is adjusted to the chosen sample volume is
underneath a carrier through which liquids can flow and which is
impregnated with a precipitating agent (4). Adjoining this is a
suitable absorptive cholesterol determining test film (6) which is
capable of taking up fluid free of precipitate from the fibre mesh.
The test film is coated on a transparent foil (7). The
determination is started by applying the sample liquid over the
carrier containing the precipitating agent. The measurement signal
which develops can be evaluated visually or photometrically from
the foil side of the construction.
This simple design is not well-suited for the investigation of
whole blood. If whole blood is to be used, then the cellular blood
components are preferably separated in a layer (3) in front of the
carrier for the precipitating agent e.g. by agents which are
described in detail in DE-A-30 29 579.
With regard to the well-known temperature dependency of enzymatic
reactions, the geometric arrangement of the test design can also be
so chosen that the cholesterol test is separated by time from the
previous reaction steps. This also allows a controlled regulation
of the temperature of the test step. A corresponding test
construction is outlined in DE-A-3130749. The preferred
construction of a rapid diagnostic agent for HDL in a test strip
form (11) is shown in FIG. 2. A fibre mesh (15), a carrier through
which liquids can flow and which contains a precipitating agent
(14) as well as a separating layer for cellular components (13) are
mounted on top of one another on a supporting foil (12). The fibre
mesh (15) protrudes from under the carriers (13) and (14) towards a
flap. The flap, which consists of a transparent foil (17) and a
test film for cholesterol (16) is attached to the supporting foil
(12) via an adhesive join (18).
The sample to be investigated is applied to the layer (13) and
flows through the layers (14) and (15), during which the non-HDL
lipoproteins are separated, into the mesh (15) under the flap. The
test reaction is started by pressing the flap with the test layer
(16) onto the layer (15). The change in colour can be followed by
means of a photometer or a reflection photometer.
In this case in comparison with the embodiment outlined in FIG. 1
lateral separating capacity of the fibre mesh is also
effective.
The method according to the present invention has other major
advantages over the known methods. It is possible to use
particularly small liquid volumes. The handling of the device
according to the present invention is very simple. Only two basic
handling steps are necessary, namely the application of a liquid
sample and the reading of a measurement after a particular time.
Other instruments are not needed apart from a suitable photometer
in the case of a quantitative determination. Transfer steps are not
needed. All types of blood, even whole blood, can be easily used
when a separating pad for cells is used first. If anticoagulants
have been added to the sample to be examined, it is recommended
that their effects on the determination be compensated. The
separation of non-HDL lipoproteins can be carried out in less than
60 seconds. The dosage of the sample volume is greatly
simplified.
Examples for the invention are given in the following:
EXAMPLE 1
Precipitating agent carrier (14 or 4) for EDTA plasma
A multifilament polyester fabric (100 .mu.m mesh size, 105 .mu.m
fabric thickness, 55 threads per cm, with 815 ul water passage per
m.sup.2 and sec) is impregnated with a solution of the following
composition:
______________________________________ Hepes buffer, 50 mM; pH 7.0
78.00 g magnesium acetate .times. 4H.sub.2 O 15.68 g dextran
sulphate (MW 50000) 2.57 g bovine serum albumin 1.78 g
______________________________________
A coating of about 57 ml/m.sup.2 is obtained.
After drying under a flow of warm air the impregnated fabric is cut
into suitable unit areas which match the corresponding test
procedure and they are integrated into the test construction.
EXAMPLE 2
Precipitating agent carrier (14 or 4) for serum
The following impregnating solution is used in a procedure
analogously to Example 1:
______________________________________ Hepes buffer, 50 mM; pH 7.0
78.00 g magnesium acetate .times. 4H.sub.2 O 10.10 g dextran
sulphate (MW 50000) 2.57 g bovine serum albmin 1.78 g H.sub.2 O
5.58 g ______________________________________
EXAMPLE 3
Precipitating agent carrier (14 or 4) for blood
A paper of suitable thickness and absorptivity e.g. tea bag paper
with a weight per unit area of 12 g/cm.sup.2 and a thickness of 50
.mu.m is impregnated with the following impregnating solution:
______________________________________ Hepes buffer, 50 mM; pH 7.0
70.20 g magnesium acetate .times. 4H.sub.2 O 4.78 g dextran
sulphate (MW 50000) 2.54 g bovine serum albumin 1.60 g H.sub.2 O
7.80 g ______________________________________
A coating of about 63 ml/m.sup.2 is obtained with this carrier.
EXAMPLE 4
Test film
A dispersion of the following composition:
______________________________________ K/Na phosphate buffer, 0.5
M; pH 7.0 17.14 g Keltrol F 0.19 g TiO.sub.2 (powder) 1.31 g
dioctyl sodium sulphosuccinate 0.40 g polyvinylpropionate
dispersion 11.70 g (50% in H.sub.2 O) diatomaceous earth (Celatom
MW 25) 17.65 g phenylsemicarbazide 0.025 g
2(4-hydroxy-3,5-dimethoxyphenyl)-4- 0.061 g
(4-dimethyl-aminophenyl)-5-methyl- imidazole-dihydrochloride
methanol 1.74 g H.sub.2 O 46.28 g cholesterol esterase 23700 U
cholesterol oxidase 6500 U peroxidase 230000 U hexanol 2.07 g
______________________________________
is prepared for the production of a reagent film in order to
quantify HDL cholesterol.
The dispersion is applied as a layer of 300 .mu.m thickness onto a
polycarbonate foil and dried with warm air. The reagent coating
which results forms a graded blue colouration with fluids
containing cholesterol depending on the cholesterol content.
______________________________________ remission R % (measurements
cholesterol in the reflection concentration photometer
Reflotron.sup.R) ______________________________________ 0 70.0 20
43.0 40 29.0 60 22.0 80 18.5 100 16.0
______________________________________
EXAMPLE 5
Fibre mesh
A mixture of borosilicate glass fibres with a fibre diameter of ca.
0.6 .mu.m and cellulose fibres with a fibre diameter of ca. 4
.mu.m, preferably in the ratio of 9:1. was used the mixture has a
weight per unit area ca. 25 g/m.sup.2 at a mesh thickness of ca.
0.21 mm; the ability of the mesh to take up water of about 370
g/m.sup.2.
EXAMPLE 6
Production of a rapid diagnostic agent (11) for the determination
of HDL
If the test construction of FIG. 2 is chosen, the following
measurements apply for the different layers:
12: 100.times.6 mm
13: 5.times.6 mm (borosilicate fleece according to DE-A-3029579,
weight per unit area ca. 60 g/m.sup.2)
14: 6.times.6 mm
15: 16.times.6 mm
16/17: 15.times.6 mm
This embodiment is suitable for a sample volume between 28-32 .mu.l
and is particularly advantageous.
It will be understood that the specification and examples are
illustrative but not limitative of the present invention and that
other embodiments within the spirit and scope of the invention will
suggest themselves to those skilled in the art.
* * * * *